Weight gain and fatty acid patterns of predators

CHAPTER 3 Taking it to the next level: trophic transfer of marker fatty acids from

3.3 Weight gain and fatty acid patterns of predators

Two of the 40 juveniles of L. forficatus (initial weight 2.3 ± 1.6 mg) escaped and two died during the experiment, all others survived. Most surviving centipedes gained weight (+19.4 ± 13.4 %). Whole FA composition of L. forficatus differed significantly between the treatments (DFA, Fig. 2, Table 1). Centipedes fed with differently nourished H. nitidus incorporated the marker FAs for the diets of the Collembola (Fig. 1c). L. forficatus fed with H. nitidus kept on C. globosum had a significantly higher amount of 18:2ω6,9 (19.1 ± 4.0

%) than when fed with H. nitidus kept on T. europaea (8.2 ± 3.8 %). When fed with Collembola kept on bacterial diets, the content of 18:2ω6,9 in L. forficatus was much lower (3.5 ± 2.2 % and 1.1 ± 1.2 %) for Collembola kept on S. maltophilia and B.

amyloliquefaciens, respectively). The ratio of 18:1ω9 to 18:2ω6,9 in centipedes fed with H. nitidus kept on C. globosum was 2.1, whereas it was 5.1 in L. forficatus fed with H.

nitidus kept on leaves of T. europaea. The bacterial marker FAs i15:0 and a15:0 were almost exclusively found in L. forficatus fed with H. nitidus kept on S. maltophilia (0.4 ± 0.5

% and 0.3 ± 0.4 %, respectively) and B. amyloliquefaciens (0.1 ± 0.1 % and 1.9 ± 0.7 %, respectively). However, they occurred in lower concentrations than in their Collembola prey. The FA a17:0, which mainly occurred in B. amyloliquefaciens, was also present in centipedes fed with Collembola kept on this diet (1.3 ± 0.7 %). Centipedes contained the C20 PUFAs 20:4ω6,9,12,15 (1.9 ± 1.9 %) and 20:5ω3,6,9,12,15 (1.3 ± 1.4 %).

-15 -10 -5 0 5 10

Fig. 2: Discriminant function analysis of NLFAs of Lithobius forficatus (Chilopoda) fed with Heteromurus nitidus (Collembola) kept on different diets (fungus, leaves, Gram-negative and Gram-positive bacteria). Ellipses represent confidence ranges α = 0.05.

Pardosa lugubris

The weight gain of Pardosa lugubris differed significantly between juvenile spiders, subadult males and subadult females (F_2,71 = 23.8, p<0.0001). Subadult male spiders (initial weight 12.5 ± 1.5 mg) lost weight during the 4 weeks of feeding (-2.0 ± 1.9 %) and subadult female spiders (initial weight 11.4 ± 3.5 mg) gained little weight (+1.5 ± 5.2 %). In contrast, juvenile spiders (initial weight 3.5 ± 1.6 mg) gained weight by 10.4 ± 6.6 %. This was also reflected in the FA patterns, with marker FAs being visible most clearly in juvenile spiders. However, in general the FA composition of P. lugubris differed significantly between treatments (DFA, Fig. 3, Table 1).

As the incorporation of marker FAs was most pronounced in juvenile spiders only these are presented here (Fig. 1d). P. lugubris fed with H. nitidus kept on C. globosum had higher amounts of the fungal marker 18:2ω6,9 (18.8 ± 1.7 %) than when fed with H.

nitidus kept on bacterial diets. However, when P. lugubris was fed with H. nitidus kept on T. europaea, the amount of 18:2ω6,9 (20.3 ± 4.4 %) was similar to that of P. lugubris fed with H. nitidus kept on C. globosum. Also, the relative leaf and fungal marker FA 18:1ω9 did not differ between these treatments. The bacterial marker FA i15:0 was only found in

spiders fed with Collembola kept on B. amyloliquefaciens (0.1 ± 0.2 %) and S. maltophilia (0.2 ± 0.1 %). The bacterial marker FA a15:0 was found in small amounts in spiders of each of the treatments; only P. lugubris fed with H. nitidus kept on B. amyloliquefaciens contained significantly higher amounts of a15:0 (1.5 ± 0.7 %) than when fed with H. nitidus kept on other diets. The bacterial FAs i17:0 and a17:0 generally were not present in P.

lugubris. All individuals of P. lugubris contained the C20 PUFAs 20:4ω6,9,12,15 (0.9 ± 0.3

%) and 20:5ω3,6,9,12,15 (1.2 ± 0.6 %) in similar amounts.

-8 -6 -4 -2 0 2 4 6

Fig. 3: Discriminant function analysis of NLFAs of Pardosa lugubris (Araneae) fed with

Heteromurus nitidus (Collembola) on different diets (fungus, leaves, Gram-negative and Gram-positive bacteria). Ellipses represent confidence ranges α = 0.05.

4. Discussion

FA profiles were transferred over three trophic levels from basal resources (fungi, leaves and Gram-positive and Gram-negative bacteria) over Collembola to centipedes and spiders, suggesting that FA analysis allows tracing the role of basal resources for predator nutrition. The transfer of marker FAs along food chains therefore may allow separation of different energy channels in soil food webs, such as the bacterial vs. the fungal channel (Moore and Hunt, 1988).

Food resources differed significantly in their FA composition, which is an important precondition for further analyses of higher trophic levels. Food resource marker FAs were present, such as 18:2ω6,9 in fungi, 18:1ω9 in leaves and the branched FAs i15:0, a15:0, i17:0 and a17:0 and 16:1ω7 in bacteria. The fungus C. globosum and leaves of T.

europaea could be distinguished by the relative proportions of 18:1ω9 and 18:2ω6,9. In T.

europaea, the ratio was more than 20 times higher than in C. globosum. The Gram-negative bacterium S. maltophilia differed significantly from the Gram-positive B.

amyloliquefaciens by the occurrence of 17:0 and cy17:0. The FA cy17:0 was postulated as a Gram-negative marker by Ruess et al. (2005). However, the percentage of cy17:0 (1.3 %) was low compared to the study of Haubert et al. (2006), who found a mean of 16.9

% and 31.4 % in the Gram-negative bacteria Enterobacter aerogenes and Pseudomonas putida. The Gram-positive B. amyloliquefaciens was characterized by high amounts of a17:0. Ruess et al. (2005) postulated branched chain FAs (iso, anteiso) as markers for Gram-positive bacteria. However, the Gram-negative S. maltophilia also contained significant amounts of the branched chain FAs i15:0 and a15:0, suggesting that these markers are specific for bacteria, but are not necessarily suitable to distinguish between Gram-positive and Gram-negative bacteria. Their abundance within total FAs likely is species-specific but may also depend on the culture medium.

Collembola feeding on the different diets retained diet specific marker FAs in their NLFAs The bacterial specific marker FAs i15:0 and a15:0 mainly occurred in H. nitidus fed with S. maltophilia and B. amyloliquefaciens, but also to a small extent in Collembola fed with leaves, possibly because the adhering phyllosphere had not been removed completely and was preferentially consumed by the Collembola. When fed with B.

amyloliquefaciens, H. nitidus additionally contained a significant amount of a17:0. In contrast, 16:1ω7, which within food sources only occurred in bacteria, was almost equally abundant in H. nitidus of each of the treatments, suggesting that it is not suitable as biomarker in Collembola. In diet switching experiments Chamberlain et al. (2005) found a gradual decrease of 16:1ω7 in Collembola and suggested that they may not be able to synthesize this FA themselves, but that the high initial abundance was caused by the previous diet of bakers yeast. Since H. nitidus in this study was also fed with bakers yeast prior to the feeding experiments, it is possible that 16:1ω7 originated from the previous diet. The Gram-negative marker cy17:0 was not detected in Collembola fed with S.

maltophilia, presumably because it only occurred in very low amounts in S. maltophilia.

The relative fungal marker FA 18:2ω6,9, occurred in significantly higher amounts in Collembola fed with C. globosum than in those fed with T. europaea. Contents of 18:2ω6,9 in Collembola fed fungi or plant litter were generally higher than in those fed with bacteria. Contents of the relative plant marker, 18:1ω9, were similar in Collembola from each of the diet treatments, suggesting that they are able to synthesize this FA

themselves. However, the ratio of 18:1ω9 to 18:2ω6,9 was twice as high in Collembola fed with leaves than when fed with the fungus, suggesting that for Collembola, this ratio is a good indicator of plant vs. fungal diets. Ruess et al. (2007) also suggested that this ratio could be used to separate herbivory and fungivory in Collembola, with low to intermediate ratios corresponding to fungal feeding and high ratios to plant feeding. Collembola additionally contained C20 PUFAs, which they are able to synthesize themselves (Chamberlain et al., 2005) which contrasts to more derived insect lineages (Stanley-Samuelson et al., 1988).

FA profiles of L. forficatus fed with the differently nourished Collembola were well separated by DFA suggesting that for these predators the FA composition of the basal resources of their prey is imprinted in their FA profile, and that dietary FAs can be traced over three trophic levels. Although their percentage decreased with trophic level, bacterial marker FAs were present in significant amounts in centipedes suggesting that they can be used to trace bacteria based food chains and energy channels in the field to at least the third trophic level. Only the FA i17:0 was not transferred to centipedes, possibly due to low amounts in Collembola or metabolic breakdown. Further, the fraction of the fungal marker 18:2ω6,9 in the diet persisted in L. forficatus and the ratio of 18:1ω9 to 18:2ω6,9 was 2.4 times higher in L. forficatus fed with Collembola kept on a leaf diet, than when fed with Collembola on a fungal diet. This suggests that the 18:1ω9-to-18:2ω6,9 FA ratio can be used to differentiate between predators living on prey feeding on plant based vs. fungal based diets, which has been proposed previously for first order consumers (Ruess et al., 2007).

The second predator, the spider P. lugubris, did not gain weight uniformly as was the case in centipedes. Gain of weight depended on initial weight and gender. Only juvenile spiders with a mean initial weight of 3.5 mg gained substantially weight, although still only half the amount of L. forficatus. Subadult male spiders even lost weight, presumably because they invested time and energy into courtship and male functions. Similarly, the weight of subadult female spiders also changed little. H. nitidus is high quality prey for juvenile lycosid spiders (Oelbermann and Scheu, 2002), whereas larger subadult spiders may prefer different prey. Further, growth rates generally decline with age and the duration of the experiment (four weeks) may have been too short for subadult P. lugubris to gain substantial weight. The unequal gain of weight was also reflected in the FA profiles, with marker FAs being visible most clearly in juveniles. Therefore, only juveniles of P. lugubris were considered for the examination of marker FAs. The bacterial markers i15:0 and a15:0 were present in spiders fed with Collembola kept on bacterial diets.

However, a15:0 was also present in small amounts in the other individuals. Presumably, these are remains of the earlier diet of these spiders in the field. The longer chained bacterial FAs i17:0 and a17:0 were not detectable in P. lugubris. Spiders may not be able

to incorporate these FAs due to a lack of proper enzymes or they may preferentially metabolize them. It was not possible to separate spiders fed with Collembola kept on fungi or leaves by the ratio of 18:2ω6,9 and 18:1ω9 since they contained equally high amounts of these FAs. For P. lugubris, the experimental period presumably was not long enough to substitute a substantial part of storage fat; therefore older FAs presumably masked effects of differing diets. Nevertheless, FA profiles of spiders differed significantly between each of the four prey treatments (Fig. 3), suggesting that FA analysis is suitable to distinguish between the different trophic chains spiders form part of in the field.

Both L. forficatus and P. lugubris contained C20 PUFAs in similar amounts as Collembola. These C20 PUFAs may either have originated from Collembola, or centipedes and spiders may have synthesized them in part themselves.

A drawback to FA analysis in the field is the lack of quantification of the dietary uptake of specific fatty acids. Future laboratory studies should aim at quantifying the contribution of dietary components to the storage fat of consumers. Considering the complexity of dietary components and the potential breakdown or conversion of marker FAs, additional methods, such as isotope labeling, may help to differentiate basal food sources in the field more precisely.

In conclusion, on the basis of two major macrofauna predators from two arthropod taxa of different phylogenetic affiliation (Chilopoda and Arachnida), this study showed that the transfer of marker FAs can be traced over at least three trophic levels. Marker FAs of bacteria, fungi and plant leaves as basal resources were incorporated via Collembola as first order consumers into predators as second order consumers. Hence, FA analysis presumably allows differentiating the trophic food chains which consumers of different trophic level form part of in the field. This is of particular importance for understanding soil food webs as it may allow differentiating between the major trophic chains in soil, i.e.

bacterial and fungal based energy channels, and therefore evaluating the relative importance of basal resources for sustaining complex food webs with energy channeled to higher trophic levels.

Acknowledgements

This study was supported by the German Science Foundation (DFG).

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Chapter 4

Fatty acid patterns as biomarker for trophic interactions: Changes after dietary switch and

starvation

Dominique Haubert, Melanie M. Pollierer, Stefan Scheu

Published in:

Haubert, D., Pollierer, M.M., Scheu, S. (2011) Fatty acid patterns as biomarker for trophic interactions: Changes after dietary switch and starvation. Soil Biology & Biochemistry 43, 490-494.

Abstract

Fatty acid (FA) analysis is becoming increasingly important for investigating trophic interactions in soil food webs. FA profiles of neutral lipids are affected by diet, and the occurrence and amount of certain FAs can reflect feeding strategies. However, to draw conclusions on feeding strategies in the field, it is necessary to know physiological parameters of fatty acid metabolism such as the detection time and storage period of FAs.

In this study we investigated the chronological change of FA biomarkers in the Collembola Heteromurus nitidus, when switched between different food sources: leaves (Tilia europaea), a fungus (Chaetomium globosum) and two bacteria (Stenotrophomonas maltophilia, Bacillus amyloliquefaciens). Additionally, we observed the change of bacterial FA biomarkers during starvation. After 14 days of food deprivation bacterial fatty acids were still detectable in a sufficient amount to use them as dietary markers. Switching diet experiments demonstrated that FAs typical for a specific diet are already present after one day and are still detectable after 14 days on a different food source, suggesting that FA analysis can integrate the food choice of Collembola over a longer period of time, in contrast to snapshot methods such as gut content analysis.

Keywords: fatty acid analysis, Collembola, food web, food source, diet, starvation, biomarker, dietary routing

1. Introduction

Lipid composition of bacteria and fungi is used as biomarker in environmental samples e.g., to quantify and classify microorganisms in soil (Tunlid and White, 1992; Frostegård and Bååth, 1996; Zelles, 1999). Only recently, fatty acids (FAs) were proposed as trophic markers for feeding strategies of soil animals (Ruess et al., 2004, 2005a; Chamberlain et al., 2005; Haubert et al., 2006). The method has been used more intensively in marine food web studies (Ederington et al., 1995; Meziane et al., 1997; Navarrete et al., 2000) and is now being applied to soil systems (Ruess et al., 2005b; Haubert et al., 2009). The method is based on the fact that it is energetically more efficient to incorporate dietary FAs directly into consumer tissue without degradation or modification (“dietary routing”), allowing to trace dietary signals in consumers by analyzing their fatty acid composition (Stanley-Samuelson et al., 1988). Therefore, FAs can be used as biomarkers for trophic links.

Collembola are widespread and abundant soil animals playing an important role in decomposition processes (Visser, 1985). In the laboratory Collembola can be reared on a wide range of different diets. However, recent measurements of stable isotopes in Collembola (Chahartaghi et al., 2005) suggest strong trophic niche differentiation in the field and very different trophic positions including detritivores, herbivores and carnivores.

However, the composition of the diet of Collembola species and its variation in time remains to be explored.

Trophic transfer of FAs from fungal food sources to nematode or Collembola grazers has been reported by Ruess et al. (2002, 2004, 2005a) and Chamberlain et al. (2004). FA biomarkers for different trophic guilds have been assigned (Chamberlain et al., 2005;

Ruess et al., 2005b; Haubert et al., 2006), i.e., i14:0, i15:0, a15:0, i17:0, cy17:0 and 16:1ω5 for bacterial feeders, high proportion of 18:2ω6,9 for fungal feeders and high proportion of 18:1ω9 and 18:2ω6,9 for species feeding on leaf litter. Additionally, the ratio of 18:1ω9 to 18:2ω6,9 has been proposed to reflect the relative contribution of fungi and leaf litter to the diet of Collembola being higher when feeding on leaves (Pollierer et al., 2010). The composition of neutral lipid fatty acids (NLFAs) in the fat body of consumers results from different processes including storage of dietary lipids, de novo synthesis, degradation and subsequent release for mobilisation to sites where they are metabolized (Beenakkers et al., 1985). However, specific FA markers for food sources in Collembola showed some robustness against variations in temperature, food quality, food deprivation

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